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The Roles of IgG, IgM Rheumatoid Factor, and their Complexes in the Induction of Polymorphonuclear Leukocyte Chemotactic Factor from Complement

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The Roles of IgG, IgM Rheumatoid Factor, and

their Complexes in the Induction of

Polymorphonuclear Leukocyte Chemotactic

Factor from Complement

Teresa Wagner, … , George Abraham, John Baum

J Clin Invest.

1974;53(6):1503-1511. https://doi.org/10.1172/JCI107700.

The induction of chemotactic factor by rheumatoid factor (RF) and by rheumatoid complexes

and their constituents was investigated. The presence of chemotactic factor was measured

by the number of polymorphonuclear leukocytes (obtained from normal individuals)

attracted through a 3 µm Millipore filter and is expressed as a “chemotactic index.” The

chemotactic index was used to measure the effect of immunoglobulins and their complexes

in stimulating production of complement-derived chemotactic factors from the sera of normal

individuals.

Two sources of IgG (F-II and DEAE-purified IgG) were used. These were prepared for use in

the native and heat-aggregated states. The same chemotactic index was obtained with both

these preparations of IgG. The chemotactic index increases when (

a

) increasing

concentrations of gamma globulin were added to a constant concentration of complement

and (

b

) when the amount of complement alone was increased.

The addition of a purified IgM RF to the mixture of IgG and complement caused a decrease

in the chemotactic index. Incubation of IgG and complement before addition of IgM RF

produced no change in the chemotactic index. The addition of a nonrheumatoid factor IgM

to IgM and complement had no effect on the chemotactic index. IgM RF and IgG alone were

not chemotactic.

These studies confirm the concept of “complement deviation” by IgM RF which occurs with,

and as a result […]

Research Article

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(2)

The Roles of

IgG,

IgM

Rheumatoid

Factor,

and

their

Complexes

in the Induction of

Polymorphonuclear

Leukocyte Chemotactic

Factor from

Complement

TERESA

WAGNER, GEORGE

ABRAHAM,

and

JoHN

BAUM

Fromthe Arthritis andClinicalImmunology Unit,MonroeCommunity Hospital, and the Department of Medicine, University ofRochesterSchoolofMedicine

andDentistry, Rochester,New York14642

ABST R AC T The induction of chemotactic factor

by

rheumatoid factor (RF) and by rheumatoid complexes

and theirconstituents was

investigated.

The presence of

chemotactic factor was measuredby the number of poly-morphonuclear leukocytes (obtained from normal

indi-viduals) attracted

through

a3Am

Millipore

filter and is expressed as a "chemotactic index." The chemotactic in-dex was used to measure the effect of immunoglobulins

and their complexes in stimulating

production

of

com-plement-derived chemotactic factors from the sera of normalindividuals.

Two sources of IgG (F-II and

DEAE-purified IgG)

were used. These were prepared for use in the native

and heat-aggregated states. Thesame chemotactic index

was obtained with both these preparations of

IgG.

The chemotactic index increases when

(a) increasing

concen-trations of gamma globulin were added to a constant

concentration of complement and (b) when the amount

of complementalonewasincreased.

The addition ofa

purified

IgM

RF to the mixture of

IgG and complement caused a decrease in the chemotac-tic index.

Incubation

of

IgG

and

complement

before ad-dition of

IgM

RF produced no change in the chemo-tactic index. The addition of a nonrheumatoid factor

IgM to IgM and complement had no effect on the chemotactic index.

IgM

RF and

IgG

alone were not

chemotactic.

These studies confirm the concept of

"complement

de-viation"

by

IgM

RF whichoccurs with, andas a result

This work was presented in part at the 36th Annual

Meeting of the American Rheumatism Association, Dallas,

Tex., 9June 1972.

Dr. Wagner's present address is: Instytut Rheumatolo-giczny, Warszawa ul, Spartanska 1, Poland.

Received for publication 29 March 1973 and in revised

form 21September1973.

of, the immune complex formation between IgG and IgM RF. The successful activation of complement chem-otactic factors by IgG may explain the synovitis with high polymorphonuclear leukocyte counts found in RF-negative arthritis.

INTRODUCTION

Previous studies have demonstrated that polymorphonu-clear leukocytes (PMN)1 obtained from patients with rheumatoid arthritis exhibited decreased chemotaxis (1). Diminished chemotaxis has also been produced by

incubation of PMN obtained from normal individuals with complexes prepared from purified rheumatoid fac-tor and gamma-G globulin (1). However, even' in those

patients with decreased chemotaxis of their peripheral

PMN, an acutely inflamed rheumatoid arthritic joint contains large numbers of PMN that have actively mi-grated into the joint. In this investigation, an attempt has been made to elucidate some factors which may be

responsible for the attraction of PMN into the synovial

fluid of the patient with rheumatoid arthritis. Since about20% of patients with clinical rheumatoid arthritis

are rheumatoid factor negative, other mechanisms (aside from complement activation by immune complex

forma-tion)

can be present which may induce the chemotaxis of PMN into the joint. In these studies, results are ob-tained with aninvitro system which shows that even in the absence of rheumatoid factor and rheumatoid factor

complexes, complement may be activated by gamma-G

globulin so that chemotactic factors are produced. Fur-ther, the inhibitory effects of rheumatoid factor on the activation of chemotactic factors will be shown.

'Abbreziations used in this paper: F-IT, fraction II;

(3)

MATERIALS

PMN were obtained primarily from a normal population of four or five laboratory personnel in whom a standard chemotactic index has been reproduced on numerous occa-sions. The sera used as a complement source for these studies were principally from two individuals. Serum from a third individual was occasionally used. All were sero-type AB and Rh negative. Blood was obtained in 50-ml

bleedings; serum was immediately removed, stored in 2-ml

portions at -70'C, and utilized within 2 mo.

Gamma

globulin

preparations

FRACTION II

Fraction II (F-II) (Difco Laboratories, Detroit, Mich.)

was used in aggregated and unaggregated forms. Before use, the F-II was dissolved in Hanks' solution (Difco

Lab-oratories) and freed of aggregates by centrifugation at

35,000 rpm for 90 min in a Beckman Model L preparative

ultracentrifuge (Beckman Instruments, Inc., Spinco Div.,

Palo Alto, Calif.). The upper two-thirds of this solution (termed "supernate") was utilized. This material was

initially checked for the presence of aggregates by the

centrifugation procedure with 'SI-labeled F-II, and then

by placing an aliquot of the supernate on a 10-40%o sucrose gradient. The gradient was centrifuged at 35,000 rpm for

15 h and separated into 30 fractions. Radioactivity in the bottom two fractions was considered to consist of aggre-gates. This comprised less than 10%o of the total

radio-activity in the gradient.

Aggregation procedure. A solution of F-II in Hanks'

medium was heated to 630C for 10 min. The resulting floc-culent was precipitated twice with 0.72 M sodium sulfate

(2). The precipitate was resuspended in Hanks' solution to the required concentration. Most aggregation was per-formed in a pH range of 6.8-7.2. In a few experiments aggregationwasperformed atpH 8.0.

Immunoglobulin G. IgG used was DEAE purified (Miles Laboratory, Inc., Miles Research Div., Kankakee, Ill.). Thiswastreatedsimilarlytothe F-II.

RHEUMATOIDFACTOR

This was prepared as previously described (3) by

im-munoabsorption of serum from a patient with rheumatoid arthritis by utilizing bromoacetyl cellulose-human IgG. The

strongly latex-positive material obtained was subjected to

Sephadex G-200 chromatography and shown to consist of

only IgM by immunodiffusion, and of a single component

by isoelectric focusing and analytical ultracentrifugation. NORMALIGM

Normal IgM was prepared by chromatography of normal human serum on Sephadex G-200 (Pharmacia Fine Chemi-cals, Inc., Piscataway, N. J.). The first peak eluted was

then further purified by DEAE column chromatography by utilizing a linear 0.015-0.15 M, pH 7.35, phosphate buffer

gradient. A latex agglutination test for anti-IgG activity

of the IgM obtained was negative. The material utilized contained no IgG or IgA by immunodiffusion.

Complement

sources

The AB Rh-negative sera used as the source of

com-plement were utilized at dilutions of 1, 10, 20, and 33A% in Hanks' solution. In these sera, IgG was present at

con-centrations of 940 and 1240 mg/100 ml of serum,

respec-tively. The "concentration of complement" in this study

referstotheserumdilution.

METHODS

In each set of experiments, two to four chambers were used for each test situation and each control. Controls

con-sisted of chambers with either sera (without added immuno-alone.

globulin or complexes) or immunoglobulin preparations The method for inducing chemotaxis of PMN, previ-ously described in detail (1, 4), is based on the method of Boyden (5) and is similar to the procedure described by Ward, Cochrane, and Muller-Eberhard (6).

Blood is obtained from normal individuals in 10-ml Vacu-tainer tubes (Becton-Dickinson and Co., Rutherford, N.J.) containing heparin. To this is added 0.75 ml of a 2%o solution of methyl cellulose in normal saline (7). After standing at room temperature for 30-45 min, the plasma layer is removed. The now enriched concentration of PMN is spun down in a Shandon Cytocentrifuge (Shandon

Southern Instruments Inc., Sewickley, Pa.) and directly

deposited on a 3-,um Millipore filter (Millipore Corp.,

Bed-ford, Mass.). The filter is immediately removed and placed

into a modified Sykes-Moore tissue culture chamber (Bellco Glass, Inc., Vineland, N. J.). Through ports in the chamber,

solutions may be injected into the upper (starting side for cell migration) or lower compartments, which are sepa-rated by the filter. Hanks' solution is usually placed in the upper compartment with the PMN. In one or two experi-ments which will be noted, serum (as a source of com-plement) was placed in the upper compartment as a con-trol. The bottom compartment was filled with the test solutions which will be described. After filling, the cham-ber was incubated for 3 h at 37'C. After incubation, the filters were removed and stained by Boyden's method (5), trimmed, and mounted on microscope slides. The cells on both sides of the Millipore filter were counted by the use of a 1-cm square ocular grid divided into 2X2-mm boxes

(8).

The chemotactic index is the number of cells counted in five of the 2-mm squares on the attractant side of the filter, divided by the number of cells on the starting side in a single 2-mm square. The dividend obtained is multi-plied by 500 to produce the chemotactic index. The fields counted were selected to avoid any areas which showed clumping of cells. 10 fields are counted on each filter.

F-II was initially used at concentrations ranging from 0.01 ,ug/ml to 10 mg/ml. Subsequent experiments were performed at concentrations of F-II and IgG from 0.001 to0.1 mg/ml.

Control experiments were performed with inactivated sera (560C for 30 min) to which the aggregated or un-aggregated F-II had been added.

Experiments were performed which compared the chemo-taxis induced by aggregated and unaggregated F-II to that obtained with DEAE column-purified aggregated or un-aggregated IgG.

Experiments were performed with prior heating of the aggregated F-II to 650C for 20 min. The F-II was sub-sequently precipitated by the sodium sulfate method (2). This was to destroy any possible enzyme activity which

might have been responsible for activation of chemotactic factors from complement. In similar experiments with

unaggregated F-II, the material was heated at 56°C for 30 min. The supernate of the centrifuged solution was used.

(4)

To rule out serum factors on the surface of the PMN,

several experiments were performed with the PMN washed

three times in Hanks' solution before centrifugation onto theMillipore filter.

F-II and IgG were combined in some experiments with IgM rheumatoid factor and IgM free of rheumatoid fac-tor activity. In all of these experiments, equal concentra-tions of IgG and IgM were used. The concentration ranges were0.01-0.1 mg/ml.

In order to investigate the deviation or inactivation of chemotactic factor, several experiments were performed with prior incubation of the serum (complement), gamma globulin and IgM rheumatoid factor in the following com-binations: (a) F-II plus complement was incubated at 370C

for 30 min. Then rheumatoid factor was added and the mixture placed in the lower compartment of the migration

chamber for the 3-h incubation period; and (b)

rheuma-toid factor plus complement was incubated at 370C for 30 min. Then F-II was added and the mixture placed in the lower compartment for the 3-h incubation period.

RESULTS

Initially the effects of aggregated and nonaggregated

gamma-G-globulin (F-II) were compared at varying concentrations. The results are seen in Table I. The concentration of the complement was held at

33j%.

Comparisonsof the chemotactic values obtained by using concentrations of F-II from 10 to 0.00001 mg/ml dem-onstrate no significant differences when unaggregated oraggregated materialwas placed in the lower chamber with complement. Studies performed with 33A% com-plement alone yieldeda chemotactic index of

363.1±47.1.

Since, as notedabove, the IgG content of the serum Used as a complement source was approximately 10 mg/ml, a concentration of added F-IIof 1 mg/ml thus onlyraises the total concentration of IgG in the chamber about20%.

The relationship of the chemotactic index to the con-centration of gamma globulin is apparent- when it is seen that chemotactic indices significantly greater than the values for serum alone are not reached until this

TABLE I

Chemotaxis of PMN with Aggregated and Unaggregated F-II

Chemotactic index (meanSEM)

Aggregated Unaggregated

With Without With Without Concentration comple- comple- comple- comple-of F-1I ment* ment ment* ment

mg/mi

10.0 640.74±158.3 - 779.5 4172.7 -1.0 620.9 464.9 - 620.0±464.9 -0.2 336.9±461.2 - 477.0±42.5 -0.1 336.2±49.0 46.8 424.0t36.4 60.0 0.01 419.9±65.3 46.0 389.3436.4 44.4 0.001 338.2-4-59.5 62.8 356.34:49.5 64.9 0.0001 254.44±77.2 38.6 228.6±443.8 62.5 0.00001 55.0±10.8 - 112.5428.6

-*Complement-33j%.

TABLE I I

Chemotaxis of PMN's with Different Concentrations of

ComplementandAggregatedF-Il

Complement Aggregated Chemotactic index concentration F-I1 (mean±SEM)

% mg/mi

33w 0 363.1±-97.1

20 0 262.5-±24.3

10 0 233.5±1=51.5 1 0 48.34-11.7 331 0.01 419.9±-65.3

20 0.01 394.04-127.6

10 0.01 223.8--56.5 1 0.01 132.4±4;17.5

concentration of added F-II (aggregated or

unaggre-gated) was reached.

Further evidence that thechemotactic factors are

pro-duced by activation of complement by the added F-II was provided. In 15 control experiments, F-II at a

con-centration of 0.01 mg/ml was added to inactivated sera (560C for 30 min). The mean chemotaxis was reduced to 86.6±+13.4. Several experiments were performed to rule out the possibility that activity of the unaggregated

gammaglobulin was due to the presence of small amounts ofaggregated material present in the preparations. The

F-II utilized was thus labeled with 'I. After labeling, it was centrifuged at 35,000 rpm and the top two-thirds of the centrifuged preparation removed. A portion was then placed on a sucrose gradient and centrifuged at

35,000 rpm at 4VC for 15 h. The gradient was collected in30 fractionsand each was counted in a gamma counter.

The percentage of radioactivity found in the bottom of the gradient averaged 9.8% of the total amount of

radioactivity. Thus, 90% of the gamma G globulin in

theseexperiments was present as unaggregated material. Since numerous concentrations of F-II were utilized,

this amount of aggregation does not appear to influence

the data, since no significant difference in activity is noted between the two forms of the gamma G globulin

(Table I).

In order to measure the effect of the serum used as

the source of complement, experiments were performed using 33A, 20, 10, and 1% concentrations of serum di-luted in

Hanks'

solution. The results are noted in Table II. Although an overall decrease is noted as the serum concentration is lowered, the most marked drop is seen

between 33A and 20% and from 10 to 1%. Most of the

studies reported in this paper were performed at serum

concentrations of 33& and 20% and are noted for the

appropriateexperiments.

(5)

re-TABLE I II

Chemotaxis of PMNatDifferent Complement Concentrations with DifferentPreparationsof Aggregated and

Unaggregated F-II and IgG

Chemotactic index (mean4SEM)

Comple-ment Aggregated

concen- IgG

tration concentration IgG* F-II1 % mg/mi

33k 0.1 289.0i60.5 336.0±42.0 0.01 295.9±44.8 419.9±65.3 0.001 462.2±4146.0

338.2±59.9

20 0.1 368.0±52.0 305.4±40.4

0.01 270.5±-88.0 -384.0±4127.6

0.001 253.3 47.0 497.2 A244.3 Unaggregated

IgG F-II

33W 0.1 434.6±69.9 424.0442.5 0.01 459.6±95.3 389.3±36.4 0.001

444.4±59.5

356.3±49.5

20 0.1 658.0±194.4 271.0±36.7§

0.01 462.5±99.4 268.0±53.6

0.001 525.8±119.9 301.1±61.5

*

IgG-Miles

Laboratories.

F-II-Difco Laboratories.

§P =0.02-all other Pnotsignificant.

lated to the chemotactic results obtained,

comparisons

were made between unaggregated and

aggregated

DEAE-purified IgG and the F-II (Difco Laboratories).

Although the F-IH utilized here is almost entirely

IgG,

wecouldnotruleoutthe effect of contaminationbyother

constituents or of preparative denaturation of the

pro-tein. The results ofthese experiments are seen in Table III.

In these experiments, IgG concentrations from 0.1 to

0.001

mg/ml

in

33A

and 20% serum were used.

Essen-tially no difference between the two preparations was

noted when the data were analyzed by statistical means.

One group of studies

(20%

complement, 0.1 mg/ml of

unaggregated

F-II and IgG) were significantly different

atthe 0.02 level. This difference between the two

prepa-rations is noted in Table III. It stands as an isolated finding, and we do not feel that this represents a true

difference between the two preparations. Control

ex-periments were

performed

with the IgG alone to rule

out the

possibility

of chemotactic activity from contami-nation by kallekrein in this preparation. At concentra-tions of 0.1-0.001

mg/ml

of

IgG,

the chemotactic

ac-'J. D. Smiley. Personal communication.

tivity was

18±3.7

with aggregated

IgG

and

24±4.3

with unaggregatedIgG.

In several experiments, the possibility was tested that the similar effects on the induction of chemotactic fac-tors by both aggregated andunaggregated F-II was due to the method of aggregation. A comparison was thus made of the chemotactic index induced by the addition

of aggregated F-II prepared in three different ways from a single batch of F-II. To destroy enzymes which

might activate the production of chemotactic factors, the aggregated material was first prepared by heating the F-II in Hanks' solution to

650C for

20 min. The

chemotactic index was

595±80.

To check pH effects,

some of this aggregated F-II was precipitated twice with 0.72 M Na2SO4 at pH 8.0, while a third group of stud-ies was performed with the sodium sulfate precipitation

at pH of 6.8. In the former, the chemotactic index was

431±89

and in the latter

experiment,

467+98. This was done to test the effects of a mildly alkaline pH on the cells or on the aggregated

F-IH.

The results demonstrate that there was no significant effect with any of the three methods of preparation of

F-IL.

The status of the PMN used in these experiments was next investigated. With the technique utilized here, the cells, after concentration with methylcellulose, are placed directly into the cell centrifuge and spun onto the Milli-pore filter. Excess plasma is removed by a filter paper pad. In this group of experiments, the cells were washed three times with Hanks' solution before placing them in the centrifuge. No differences were noted between un-washed and un-washed cells. This mitigates against the ef-fects of any cell-bound globulin or complement compo-nents from the cell donor affecting the results.

A series of experiments was then performed to look at the activation of chemotactic factors by using the

complex formed from rheumatoid factor and IgG.

Vari-ous experiments were performed which involved prior

FIGURE 1 Interrelationships of IgG, IgM rheumatoid

fac-tor (RF), and normal IgM in the induction of chemo-tactic factorsfromcomplement (C).

(6)

incubation of one or both immunoglobulin components with complement and each other. A number of experi-ments were also performed tocompare a nonrheumatoid factor IgM with the IgM rheumatoid factor. In Fig. 1

the data are displayed so that the relationship of the results to the different test conditions can be readily

observed. In the left-hand corner is noted the expected result of the reaction of complement with a concentration of F-II at0.01 mg/ml. When rheumatoid factor is added to the other components, there is a significant drop in the chemotactic index to 162.7. This value (shown in the center) demonstrates the inhibitory effect of rheu-matoid factor on the activation of chemotactic factor. If, however, the F-II and complement are preincubated and the rheumatoid factor then added (center left), a

chemotactic index of 243.7 is obtained. This value is

significantly higher than that reached when the rheuma-toid factor was initially present with the F-II and com-plement. Since activation of complement takes place before addition of the rheumatoid factor, the value

obtained is in the same range as that for the F-II and complement alone.

The deviation of complement (reduction of comple-mentactivation) by the rheumatoid factor is also seen in two further experiments. At the top center of the figure,

results are shown in which rheumatoid factor and com-plement are preincubated and then F-II is added. A chemotactic index of 110.4isobtained. Thisdemonstrates that the rheumatoid factor inhibited the production of

chemotactic factor with deviation of complement

ac-tivation, since addition of theF-IIdidnot induce chemo-taxis. This idea is further substantiated by comparison of the index in this experiment with that shown in the lower right where a similar chemotactic index was ob-tained with rheumatoid factor and complement alone,

i.e., without addition of F-II. This same degree of in-hibition of complement activation is also shown in the experiment in the upper right. Here, the

ability

of the F-II to induce complement activation was blocked by

its prior incubation with rheumatoid factor.

The chemotactic indices shown in parentheses in the top and right boxes are those values produced when non-rheumatoid factor IgM was used instead of the rheuma-toid factor. While only small amounts of material were available for a few experiments in each case, it is nevertheless apparent that deviation of complement did

not occur with this material. It further shows that the

effect of the rheumatoid factor appears to be specific

in each case where it reacted with either complement or

F-IH.

In several experiments, the purified rheumatoid factor

was added to the IgG prepared by column

chromatog-raphy. This was done to demonstrate that a

preparation

of

IgG

of greater

purity

would give results similar to

those seen when utilizing F-IH. The same degree of complement deviation was seen with both preparations.

The chemotactic index of rheumatoid factor plus F-IH

andcomplement was 162.7, while with the purified IgG

with rheumatoid factor and complement, the chemotac-tic index was 155.9. This demonstrates the same degree

ofreactivity of the rheumatoid factor with both prepara-tionsof IgG.

DISCUSSION

The preeminent hypothesis for the cause of the synovitis -of rheumatoid arthritis considers the inflammatory stim-ulus to be the complex formed by rheumatoid factor

(IgG and/or IgM) and gamma globulin (IgG) (9).

The formation of this complex presumably triggers com-plement activation, which results in the production of chemotactic factors (C3a, C5a, C567). These attract PMN into the joint, where they phagocytose the com-plexes (10). The lysosomal enzymes released (11) eventually lead to destruction of the joint. However, this theory does notaccount for those patients in whom rheumatoid factor is not present and in whom synovitis andinflammation can be as severe as that found in rheu-matoid factor-positive patients (12).

Hedberg, in a comparison of synovial fluids from fac-tor-negative and factor-positive patients, showed no

dif-ference in the total white blood cell count of the synovial fluid (13). This was also noted previously by Hollings-worth, Siegel, and Cressey (14), who found the per-centage of PMN to be as high in the synovial fluid of

factor-negative patients as in those patients in whom rheumatoid factor is present. In addition, it has been shown that theIgG concentration in synovial fluids from these two groups is similar and indeed higher than in any other form of arthritis (13, 15). Smiley, Sachs, and

Ziff (16) found the major immunoglobulin produced by explants from synovial tissue was IgG. Therefore,

an-other mechanism, possibly related to the presence of

IgG, might account for the synovitis and large numbers of PMN found in the synovial fluid of patients with

factor-negative rheumatoid arthritis.

Furtherconfirmation of the similarities in thesynovial

fluids from factor-positive and factor-negative patients

is found in the studyby Zvaifler (17) of the breakdown product of C3 in these two groups. To account for the presence of this product, the reaction of aggregated

gamma globulin with rheumatoid factor is postulated.

However, in his studies there was an equally significant number of rheumatoid factor-negative patients in whose

synovial fluids this material was found. These data are

consistent with the experimental results found in our

studies.

The importance of

IgG

in rheumatoid arthritis is

(7)

(18), who looked at fluorescent staining in the skin and

vessels of patients with rheumatoid arthritis, comparing

them to controls. They found a significant difference in the localization of IgG and IgA in the walls of small

cutaneous vessels. These workers point out that earlier studies also showed IgG localized in the small vessel walls in rheumatoid arthritis in synovial tissue and skin. It should be noted that they found no difference in the

deposition of IgM between the rheumatoid arthritis and controlsubjects.

Restifo,Lussier, Rawson, Rockey, and Hollander (19)

have suggested that inflammation may be produced by

the injections of 7S gamma globulin into an inactive

joint of patients with rheumatoid arthritis. Although

complex formation was felt to be responsible for the production of the synovitis, several interesting points

are noted in their study. The highest white blood cell count reported was found in a joint injected with

un-aggregated gamma globulin where the synovial fluid had the lowest latex titer (1: 40) of any in the study. Subsequent

investigations

by Hollander, Fudenberg,

Rawson, Abelson, and Torralba (20) confirmed the

marked effect of unaggregated gamma globulin from rheumatoid arthritis patients on the induction of an

in-flammatory reaction in the joints of rheumatoid factor-positive patients. In one experiment in which nonrheu-matoid gamma globulin was used, the effect of the

un-aggregatedgamma

globulin

wasslightly better in induc-ing the cellular response. These in vivo studies support the biological activity of unaggregated gamma globulin

wehave found in our invitro experiments. It should be noted thatSliwinski andZvaifler (21) in similar

experi-ments were unable to reproduce joint inflammation with the injection of aggregated gamma globulin, although

one

patient

responded to the injection of unaggregated

gamma globulin with a marked inflammatory response

(which

wasnotreproducible).

Aggregate-free IgG has been shown to fix comple-ment. Runge and Mills (22) showed that aggregate-free gamma

globulin

fixed complement in 5 of 11 patients,

and the fixation found in several was up to

30%.

No difference was found between native or aggregated

au-tologous IgGwhen they performed skin tests in rheuma-toid arthritis

patients. Augener, Grey, Cooper,

and

Muller-Eberhard atLa

Jolla

have also demonstrated the binding of monomeric

IgG

with Cl. This binding was

less than that which took place after aggregation of the

same native

subgroup

of

IgG (23).

Normansell (24) found no convincing evidence that

rheumatoid factor reacts more strongly with altered

gamma G globulin than with native gamma G globulin.

It is clear that the results we have obtained, which show

no

difference

between

heat-aggregated

gamma G

globu-lin and unaltered gamma G

globulin,

are akin to a

sub-sequent demonstration by Normansell

(25)

that the binding of rheumatoid factor to heat-aggregated gamma

G globulin was not significantly stronger than that to

unaltered gamma G globulin. Schur and Kunkel

(26)

also found that rheumatoid factor would react with

un-aggregated gamma globulin. Our results are consistent

with the data in these studies (Fig.

1).

In the work of Hurd, LoSpalluto, and Ziff (27), it was feltby the authors that the inclusions found in

nor-mal PMN after the addition of IgM rheumatoid factor to rheumatoid factor-negative synovial fluids was due

to the reaction of the added rheumatoid factor with al-tered gamma globulin present in the synovial fluid.

However, they could not otherwise demonstrate the presence of aggregated gamma globulin. Since as

indi-cated above, Normansell

(25)

and Schur and Kunkel

(26) showed rheumatoid factor could react with unag-gregated gamma globulin, there may not have to be a

requirement for aggregation of the gamma globulin in thesynovial fluid to produce the complexes.

Numerous workers have found this same phenomenon in varying degrees. Allen and Kunkel (28) called atten-tion to an autospecific rheumatoid factor which had strong affinity for native gamma globulin. They also

re-ported this in gamma myeloma proteins. Oreskes and

Shore (29) showed that there was an inhibitory effect

of added native gamma globulin on agglutination titers. Although the effect was not as marked as with the same gamma globulin aggregated by heating, there was a

sig-nificant effect with the native gamma globulin. Han-nestad showed in a number of experiments, that native gamma globulin was firmly bound to rheumatoid factor and demonstrated clearly that this was not aggregated gamma globulin (30). He felt that when this reactive complex was present in sera, it represented the hidden rheumatoid factors described by Allen and Kunkel (28). The complex could be inhibited by low concentrations of native gamma globulin. In these studies he was able

to show that 7S gamma globulin combined firmly with

isolatedgamma M rheumatoid factor. This complex was

accompanied by similar sedimentationproperties and the

same inhibition of rheumatoid factor activity as was noted in whole sera. Labeled 7S gamma globulin was shown in sucrose density studies to be found with the

macroglobulin rheumatoid factor.

The similarity of effects with both the unaggregated and aggregated gamma globulins and their reactions

with the rheumatoid factor used in this experiment has also been demonstrated by Pike and Schultz (31). They studied the inhibitory effect of several types of gamma globulin on the titer or rheumatoid arthritis sera in the sensitized sheep cell test. They found in one rheumatoid arthritis sera the same 16-fold decrease in titer with un-heated human gamma globulin as with human gamma

(8)

globulin that had been heated at 630C for 10 min. The results seen in our experiment are consistent with their

findings and would indicate that the similarity of results with aggregated and unaggregated gamma globulin may

be more likely due to the particular rheumatoid factor used in the studies. This was also shown by Allen and Kunkel (28) and indicates our rheumatoid factor prob-ably represents a factor with a high specificity for na-tive unaltered gamma G globulin.

In our in vitro experiments we have shown evidence that leads to a hypothesis for the role of IgG as the principle factor responsible for the chemotaxis of PMN into the joint of the rheumatoid factor-negative, as well as the rheumatoid factor-positive, patients. Indeed it appears that rheumatoid factor, by preventing the ac-tivation of chemotactic factors, may be playing a pro-tective role. This blocking effect of rheumatoid factor

on complement activation has been pointed out by sev-eral groups (32-34). Schmid, Roitt, and Rocha (32) related this effect to the concentration of IgG in the reaction system. Our control experiments with nonrheu-matoid factor IgM show this inhibition to be a property of the rheumatoid factor activity of the macroglobulin. This "deviation" of complement by rheumatoid factor

wasavoided by prior incubation of IgG with complement

(Fig.

1).

Evidence for the blocking activity of IgMantibody in complement fixationhas been shown for other IgM

anti-bodies beside rheumatoid factor. Scott and Russell (35)

recently demonstrated a similar type of blocking of

complement fixation with antidengue IgM antibody. The blocking effect of rheumatoid factor has also been

found in asystem wherethrombocyte isoantibodies were

blockedby theaddition of rheumatoid factor (36). In early rheumatoid arthritis there is apparently little relationship between the presence of large numbers ofPMN and rheumatoid factor in synovialfluid. In fact, inastudybySchumacher and Kitridou

(37),

the patient with the highest titer of rheumatoid factor in the syno-vial fluid had one of the lower leukocyte counts, with a low percentage of PMN. In the two patients with the

highest leukocytecount

(15,000

and

20,000),

onehadno

rheumatoid factor and the other had a minimal amount.

Both of these patients also had the highest percentage of PMN. This is of interest in the view of our finding

that in the presence of rheumatoid factor, we found a

decrease in chemotaxis (Fig. 1).

Since we found that adding IgG (increasing the con-centration in the chamberby

20%

or more) producedan increase in chemotaxis, it is apparent that the number of PMN in the

joints

of patients with rheumatoid

ar-thritis may have some relationship to the high levels of gamma globulin found in the synovial fluid of patients

with this disease, whether rheumatoid factor is present

or absent.

Further support for the importance of gamma

globu-lin in joint inflammation comes from the study of

Cham-berlain, Shapland, and Roitt (38), who showed that autologous heat-aggregated IgG, injected intradermally

in patients with rheumatoid arthritis and in controls,

produces an "Arthus-type" reaction in the patients.

However, their studies differ from our results because the skin reaction they produced with heat-aggregated

material was significantly greater than with unaggre-gated IgG.

The chemotactic activity in serum alone (which serves as the source of complement) is probably due to the presence ofIgG reaction with the complement. Albumin has been 'shown not to be chemotactic (39). Kinsella, Baum, and Ziff (40) found morphological evidence for the association of IgG and complement factors in their appearance in a diffuse cytoplasmic staining pattern in the A and C cells of synovial tissue. The same degree of

fluorescent staining was seen in rheumatoid factor-posi-tive and rheumatoid factor-negafactor-posi-tive patients.Evenin cells from rheumatoid factor-positive synovial tissue, this as-sociation was found without staining for IgG and IgM

rheumatoid factors. This was confirmed by the studies of

Bonomo, Tursi, Trizio, Gillardi, and Dammacco, (41) who found similar staining patterns for IgG and com-plement in synovial cells.

In a recent article on the pathogenesis of joint in-flammation in rheumatoid arthritis, Zvaifler (42) postu-lated the activation of chemotactic factors by immune complexes to account for the "remarkable accumulation of neutrophiles in rheumatoid synovial effusions.. On the basis of our investigation, we believe the induc-tion of chemotactic factors from complement can occur

in the joint in the absence of complex formation. We

feel that the clinical status of the joint inflammation in rheumatoid factor-negative patients with accumulations of PMN like thatseen in factor-positive patients is more related to the similarly elevated levels of IgG found in theirsynovial fluids. However, the presence of complexes and their ingestion bythe PMN and phagocyticsynovial cells causes an outpouring of lysosomal enzymes into the synovial fluid. This, then, can explain the well-known difference in the pathophysiology between these

two groups of patients-the factor-positive

complex-forming group of patients show more erosive disease (43). Even if the IgG is altered or aggregated in the rheumatoid factor-negative patients, it apparently will have little or no effect on the release of lysosomal

en-zymes from PMN.

From these data, it seems probable that gamma

(9)

percent of large numbers of leukocytes in the synovial fluid. It has been well demonstrated that factor-negative rheumatoid arthritis does not lead to the degree of joint destruction seen with factor-positive arthritis. It has also been shown that lysosomal enzyme titers are lower in the synovial fluid in factor-negative arthritis (44). This would indicate that joint damage is more likely to appear when complexes can be ingested and cause leak-age of lysosomal enzymes into the synovial fluid. The mere presence of thePMN in high concentrations thus is not sufficient to lead to high lysosomal enzyme levels and joint destruction. On the basis of our studies, it is apparent that rheumatoid factor complex formation is not necessary for theinflux of PMN into the joint, but may theoretically inhibit this process to some extent.

ACKNOWLEDGMENTS

We are indebted to Dr. Peter Ward for his advice during

the course of this study. We wish to thank Mts. Charene

Winney for expert technical assistance and Mrs. Maureen E. Laffin for her skillful preparation of this manuscript.

'This study was supported in part by a grant from the Monroe County Chapter of the Arthritis Foundation.

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(10)

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References

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